Summary: The HIV envelope gp41 is a heavily glycosylated transmembrane protein. The ectodomain region, located on the outer surface of the viral membrane, directly mediates membrane fusion events via an N-terminal fusion peptide (FP). The structure of a truncated gp41 ectodomain (residues 27-154) lacking FP and the C-terminal membrane anchor is a rod-like trimer comprising three parallel N-terminal alpha-helices. These are assembled as a coiled-coil in the center with three antiparallel C-terminal alpha-helices packed on the outside with highly flexible loops connecting the inner and outer helices. This 6-helical bundle (6HB) is the fusion-active conformation. There have been no direct structural determinations on the pre-fusion conformation where the N- and C-terminal helical regions are extended (pre-hairpin intermediate). The blocking or inhibition of the transition from the pre-hairpin to 6HB conformation is a major target for anti-HIV drug targeting. To understand interactions of gp41 domains associated with lipid membranes, constructs were made that included the N-terminal FP and the membrane anchor. This membrane protein (HIV-1 gp41 residues 1-194) was expressed in bacteria and purified as a protein-detergent complex. In the complex, detergent acted as surrogate lipid to stabilize the protein conformation. High resolution nuclear magnetic resolution (NMR) techniques were used to probe the structure, revealing a high degree of intrinsic mobility for the trimeric protein complex. The fusion peptide exhibits high amplitude motion on the fast nanosecond time scale and the linker between the N- and C-terminal helical regions shows both fast and slow (microsecond) motions. These findings are compatible with the protein switching rapidly between the pre-hairpin intermediate, three-helical bundle state, and the late-fusion, anti-parallel 6HB. A gp41 construct (residues 27-194) was also used where the fusion domain was removed in order to improve protein expression and solubility. In this construct the N-terminal domain appears tightly associated in a trimeric conformation similar or close to that in the 6HB, whereas the C-terminal domain transitions between extended (pre-fusion) and closed (6HB fusion-active) conformations. Hence, both the full length gp41 (1-194) and the shorter gp41 27-194 constructs appear to be pre-fusion structures of gp41. In biological terms, the pre-fusion state exists before the viral and cell membrane make contact and fuse, which presents a potential new target for fusion inhibitors and is analogous to gripping HIV at its weak point. The NMR studies are being extended to include gp41 constructs incorporated into artificial lipid membranes (liposomes) to simulate more physiological conditions. With the current improvements made in the protein preparations, we are also reexamining conditions for protein crystallization especially of lipid-protein complexes. These structural studies will provide additional insight into the fusion mechanism and potential targets for anti-HIV intervention. The central gp41 ectodomain (6-HB) has been studied extensively and is a homotrimer. Therefore, gp41 was considered to remain homotrimeric during all stages of the fusion process. A very recent NMR structure of the gp41 transmembrane domain (TM) alone showed it can form trimers. Our previous NMR and sedimentation equilibrium studies of gp41 (published in Structure in 2014 and mentioned above) indicated, however, a monomer-trimer equilibrium that may have functional importance during the fusion process. We have established by fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) that gp41 with the TM embedded in dodecyl phosphatidyl (DPC) micelles undergoes reversible trimeric self-association with dissociation constant in the low micromolar range. This is similar to our previous results using analytical ultracentrifugation. The monomeric potential of the TM, especially at early stages of the fusion process, may play an important mechanistic role during the transition from an elongated gp41 pre-fusion intermediate, bridging viral and host-cell membrane, towards the post-fusion six-helical bundle conformation. A gp41-targeted fusion inhibitor must interfere with this transition and monomeric, partially monomeric or trimeric states all present potential binding epitopes. Based on this premise, gp41 self-association is a valid drug target model and the florescence spectroscopy mentioned above may have potential as a high-throughput assay system that could be used to screen drug libraries. This work has been submitted for publication (July 2016). Ongoing work using election spin resonance (ESP) is examining the dynamics of gp41 movement, including the movement of specific domains during the trimerization process.